Alzheimer's disease (AD) is an age-related brain degenerative disease that is the most common cause of intellectual failure in late life. Neuritic or senile plaques and neurofibrillary tangles (NFT) are the hallmark characteristic of the histopathology of Alzheimer's brains. These plaques and tangles are believed to result from deposits of two different proteins which share the properties of the amyloid class of proteins specific for AD.
The major protein component of amyloid is an .sup..about. 4 kilodalton (kd) protein, designated the beta-protein or A4 protein due to a partial beta pleated structure or its molecular weight, respectively. The amino acid sequence of A4 has been defined (Wong et al., (1985) Proc Natl Acad Sci USA 82:8729-8732) and full-length cDNA encoding a primary translation product of 695 residues has been cloned (Kang et al., (1987) Nature 325:733-736) while other cDNAs have been identified which encode a 751-residue or 770-residue precursor form (Ponte et al., (1988) Nature 331:525-527; Tanzi et al., (1988) Nature 331:528-530; and Kitaguchi et al., (1988) Nature 331:530-532).
The A4 protein accumulates extracellularly, both in brain parenchyma and in the walls of blood vessels, generally as amyloid plaques which form aggregate fibril structures and are insoluble on SDS-polyacrylamide gels. The fibrils are generally identified as amyloid based on their green birefringence after staining with Congo red and their 40- to 90-A diameter.
The second protein, mentioned previously, accumulates intracellularly in neurons of Alzheimer's brains (Castano and Frangione, (1988) Lab Invest 58:122-132) and forms tangles composed of structures resembling paired helical filaments (PHFs). In contrast to the beta-amyloid protein, the primary structure and number of proteins comprising PHFs are unknown. PHF-containing neurites are found in the periphery of the plague, whereas deposits of beta-amyloid protein form the central core of mature plaques, surrounded by degenerated neurites and glial cells.
Although the etiology of AD is unknown, it has been demonstrated that the frequency of neuritic plaques found in the cortex of AD patients correlates with the degree of dementia (Roth et alo., (1966) Nature 209:109-110; Wilcock ad Esiri, (1982) J Neurol Sci 56:343-356). The therapeutic goals in amyloidosis are to prevent further deposition of amyloid material and to promote or accelerate its resorption. To date, there are no means available to treat the pathogenesis of AD and the paucity of understanding concerning the mechanism of amyloid formation in AD is a major obstacle in the development and design of therapeutic agents that can intervene in this process. Moreover, no animal models for brain amyloidosis with beta-amyloid protein deposits or PHFs exist, creating yet another obstacle to test such putative therapeutic agents.
Logical therapeutic approaches are now, however, emerging for treating the particular amyloidosis associated with AD. These approaches are attributable, in part, from the successes and failure gained in attempting to treat other forms of amyloidosis, such as the use of dimethyl sulfoxide which blocks amyloid formation from Bence Jones proteins in vitro (Coria et al., (1988) Lab Invest 58:454-458) and use of colchicine to reduce the size of renal amyloid deposits and induce clinical remissions in several cases of familial Mediterranean fever and amyloid nephropathy (Ravid et al., (1977) Ann Intern Med 87:568-570).
Efforts directed to the design of in vitro models of age-related cerebral amyloidogenesis using A4-derived synthetic peptides are disclosed in Castano et al., (1986) Biochem Biophys Res Comm 141:782-789, and in Kirschner et al., (1987) Proc Natl Acad Sci USA 84:6953-6957. Castano et al. demonstrated that amyloid fibrils could be formed in vitro when using a synthetic peptide corresponding to the amino-terminal 28 residues of the amyloid core protein. This 28 residue peptide, as well as a 17 residue sequence contained within the 28 amino acids, both formed fibrils which stain similarly to material a 17 residue seqeunce contained within the 28 amino acids, amyloid fibrils were soluble, unlike the naturally occurring insoluble amyloid isolated from Alzheimer's brains. Kirschner et al. demonstrated that the same 28 residue peptide could be produced as an insoluble aggregate; however, this particular in vitro model is not expected to correlate well to the cellular environment in which amyloid deposition occurs.
Dyrks et al., (1988) EMBO J 7:949-957 showed that a shortened cell-free translation product comprising the amyloid A42 part and the cytoplasmic domain of the 695-residue precursor can form multimers. While aggregation was observed employing an in vitro cell-free system, this system fails to reveal whether aggregation of the translation product would naturally follow in vivo. Moreover, the in vitro cell-free system does not address protein stability issues, that is, whether adequate levels of the protein could be expressed, whether protein proteolysis exists, and other concerns generally associated with in vivo expression of recombinant proteins.
Therefore, there exists a need for a definitive cellular deposition model with which one may assay agents capable of chemically intervening in the process of amyloid deposition. Such a method should be relatively simple to perform and should be highly specific in distinguishing AD plaques from the plaques associated with other disorders. Furthermore, it is desirable that the assay be capable of being reduced to a standardized format. The present invention satisfies such needs and provides further advantages.